New azodyrecins identified by a genome mining-directed reactivity-based screening

Only a few azoxy natural products have been identified despite their intriguing biological activities. Azodyrecins D–G, four new analogs of aliphatic azoxides, were identified from two Streptomyces species by a reactivity-based screening that targets azoxy bonds. A biological activity evaluation demonstrated that the double bond in the alkyl side chain is important for the cytotoxicity of azodyrecins. An in vitro assay elucidated the tailoring step of azodyrecin biosynthesis, which is mediated by the S-adenosylmethionine (SAM)-dependent methyltransferase Ady1. This study paves the way for the targeted isolation of aliphatic azoxy natural products through a genome-mining approach and further investigations of their biosynthetic mechanisms.


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. Functional annotation of ady in Streptomyces sp. RM72 Table S2. Functional annotation of ady in Streptomyces sp. A1C6   General remarks. 1 H and 13 C NMR spectra were recorded on a JEOL ECA500 spectrometer (500 MHz for 1 H NMR), a JEOL ECX400P (400 MHz for 1 H NMR), a JEOL ECS400 (400 MHz for 1 H NMR), a JEOL ECZ400 (400 MHz for 1 H NMR) or a Bruker AVANCE Neo (500 MHz for 1 H NMR) spectrometer. Chemical shifts are denoted in δ (ppm) relative to residual solvent peaks as internal standard (CD3OD, 1 H δ 3.31, 13 C δ 49.0, DMSO-d6, 1 H δ 2.50, 13 C δ 39.5). Electrospray ionization mass spectrometry (ESI-MS) spectra were recorded on a Thermo Scientific Exactive mass spectrometer. Liquid chromatography-mass spectroscopy (LC-MS) experiments were performed with a Shimadzu HPLC prominence system coupled with a Shimadzu LCMS-2020 spectrometer or an amaZon SL-NPC system (Bruker Daltonics). All reagents were used as supplied unless otherwise stated. Escherichia coli DH5α was used as a host for general cloning. Oligonucleotides used for genetic manipulation were purchased from Fasmac Co.

Supplementary figures
N2H4-detecting reactivity-based screening. 50 L of crude extracts of actinobacteria were mixed with an equal volume of assay solution containing 10 mM p-(dimethylamino)benzaldehyde and 1 M HCl, and incubated at room temperature for 10 min. The resultant mixture was diluted with an equal volume of methanol, centrifuged at 20,630g for 10 min, then the supernatant was analyzed by Shimadzu HPLC system equipped with SPD-M20A. 5 L of the reaction mixture was loaded onto COSMOSIL 5C18-MS-II 2.0 × 100 mm (nacalai tesque). The sample was eluted by H2O/MeCN containing 0.1% formic acid with a linear gradient: 2-98% for MeCN + 0.1% formic acid over 5 min with a flow rate at 0.4 mL/min. Column eluates were monitored by UV absorption at 485 nm.
Isolation of azodyrecins. Streptomyces sp. RM72 and Streptomyces sp. A1C6 were cultured on YMS ++ solid media (0.4% yeast extract, 1.0% malt extract, 0.4% soluble starch, after pH was adjusted to 7.4 with KOH solution, 2.0% agar was added. 10 mL of 1 M MgCl2 and 8 mL of Ca(NO3)2 were added after autoclaving) and SFM solid media (2.0% mannitol, 2.0% soya flour, 2.0% agar), respectively, at 30 °C for 7 days. The resultant media was extracted by methanol, then the residual agar pieces were removed by filtration. Solvents were removed from filtrate, and residues were partitioned between H2O and ethyl acetate. The organic layer was further partitioned by hexane, then subjected to HP20 column. The column was eluted by a gradient mixture of H2O/MeOH, then the fractions that generate N2H4 upon acid hydrolysis were combined and subjected to silica gel chromatography (40-50 μm silica gel 60N (Kanto Chemical Co.). The column was eluted by a gradient mixture of hexane/CHCl3 then the fractions that generate N2H4 upon hydrolysis were combined and the solvent was removed. The crude sample was separated by HPLC with COSMOSIL 5C18-MS-II  Cytotoxic assay. In a manner analogous to the previous report, 1 the cytotoxic activities of compounds against human ovarian adenocarcinoma SKOV-3 cells, malignant pleural mesothelioma MESO-1 cells, and immortalized T lymphocyte Jurkat cells, were examined. SKOV-3 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 μg/mL). MESO-1 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 μg/mL). Jurkat cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, penicillin (50 U/mL), streptomycin (50 μg/mL), and GlutaMAX. All cell lines were seeded in a 384-well plate at a density of 1000 cells/well in 20 μL of media and incubated at 37 °C in a humidified incubator with 5% CO2. After 4 h, 2-fold serial dilution samples dissolved in DMSO were added to the cell cultures at the concentration of 0.5% (0.1 μL) and incubated for 72 h. Cell viabilities were measured using a CellTiter-Glo luminescent cell viability assay and EnVision multilabel plate reader.

S4
P388 murine leukemia cells were cultured in DMEM, supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum in 5% of CO2 cell incubator at 37 ℃. The cells were placed a 96-well cell culture plate at a density of 1 × 10 4 cells/well, then 1 µL of test solution in various concentrations (samples were dissolved in DMSO) added to cell plates and incubated for 48 h.
Doxorubicin hydrochloride was used as a positive control. Finally, 50 µL of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (1 mg/mL dissolved in PBS buffer) were added to each well and the plates were incubated for 4 h. After the medium was removed, the precipitated dye was solubilized by DMSO, and measured by a microplate reader with the absorbance at 570 nm.

S5
Preparation of demethylazodyrecin E (11). Demethyl azodyrecin E (11) was prepared by treating azodyrecin E (8) with 2 M NaOH aq. at room temperature for 20 min. The reaction mixture was acidified with 2 M acetic acid aq. until pH 7, extracted with EtOAc (3 times), and the solvent was  5 Protein-coding regions were predicted by prodigal (2.6.3). 6 Publicly available data were retrieved from NCBI database by using efetch (16.2) and NCBI dataset (11.22.0).
To assess the distribution of VlmA-like enzymes in publicly available database, protein sequences of   The plates were extracted by methanol for overnight at room temperature then debris was filtered off.
The filtrate was evaporated, and the residue was dissolved in 5 mL methanol. 50 L of the supernatant were subjected to the N2H4-based reactivity assay following the procedure described in the previous section.